Thrombospondin is a multi-functional protein capable of interacting with numerous macromolecules, e.g. fibronectin (Lahav, et al., 1984, Cell 31:253-262; Lahav, et al. 1984, Eur. J. Biochem. 145:151-156), heparin (Lawler, et al. 1981, Thromb. Res. 22:267-279) and collagen (Lahav, et al. 1984, Cell 31:253-262, Mumby, et al., 1984 J. Cell. Biol. 98:646-652). All of these molecules are constituents of the extracellular matrix, suggesting that thrombospondin forms complexes with other matrix components following its deposition into the matrix.
Gelder and Brown reported that thrombospondin inhibits interactions of fibronectin with gelatin (Gelder, F. B. and Brown, S. T. 1987, J. Lab. Clin. Med. 110:548-557). Platelet thrombospondin and an unidentified but biologically similar plasma protein were shown to inhibit the gelatin-binding activity of fibronectin; however, the domains or sequences of thrombospondin responsible for the interaction remained unknown. The interactive region has been implicated to be a different site on fibronectin, than the fibrin binding domain (Homandberg, G. A. and Kramer-Bjerke, J. 1987, Thromb. Res. 48:329-335) and at least two distinct domains of thrombospondin have been shown to bind fibronectin (Dardik, R. and Lahaw, J. 1989, Eur. J. Biochem. 185:581-588). Fibronectin and heparin compete for binding to the 27 kDa fragment of thrombospondin suggesting that these two proteins share a common or closely oriented binding site within the N-terminal domain of thrombospondin. Thrombospondin has also been shown to disrupt focal contact adhesions of endothelial cells attached to a fibronectin matrix (Murphy-Ullrich, J. E., and Hook, M. 1989, J. Cell. Biol. 109:1309-1319). The mechanism of this effect is unknown, but inhibition of the activity of thrombospondin by sulfated polysaccharides suggested that the heparin-binding domain of thrombospondin is involved.
A number of biologically active peptides from thrombospondin have been identified and isolated. Two peptides, ArgGlyAspAla (Lawler, J. and Hynes, R. O. 1986, J.Cell Biol. 103:1635-1641) and ValThrCysGly (Prater, et al., 1991, J.Cell Biol. 112: 1031-1040) are proposed to be ligands for the interaction of thrombospondin with protein receptors on the cell surface. The thrombospondin peptide TrpSerProTrpSer (Guo, et al. 1992, Proc. Nat. Acad. Sci. USA, 89:3040-3044) binds to heparin and sulfatide. However, there are no known thrombospondin peptides that are capable of binding to fibronectin or related collagen binding proteins.
Fibronectin has been implicated in a variety of cell contact processes, including cell attachment and migration. Fibronectin interacts with collagen through the "gelatin binding domain" of fibronectin and this interaction between collagen and fibronectin is fundamental to the organization of extracellular matrices and the behavior of these cells on substrates (Vaberi, et al., 1978, Proc. Natl. Acad. Sci. USA, 75: 4944-4948). Fibronectin is essential for the attachment and migration of many cells, including various tumor and cancer cells.
Accordingly, there is a need for inhibitors of fibronectin that will bind to fibronectin or related collagen-binding proteins with high affinity. There is particularly a need for such inhibitors which will bind to fibronectin or related collagen-binding proteins to prevent the fibronectin-dependent cell adhesion to collagen matrices and to inhibit interaction with collagens of other proteins that share homologies with the gelatin-binding domain of fibronectin.
It is, therefore, an object of the present invention to provide highly effective peptides having sequences which bind specifically and with specific affinity to fibronectin or other proteins having homologies with the fibronectin gelatin-binding domain so as to inhibit fibronectin-dependent cell adhesion to collagen matrices and to inhibit interaction of other proteins having homologies with the fibronectin gelatin-binding domain with collagens.
It is a further object of the present invention to provide pharmaceutical compositions containing at least one peptide which has a high affinity to fibronectin or other protein having homologies with the fibronectin gelatin-binding domain.
It is yet another object of the present invention to provide a method for binding fibronectin or other proteins having homologies with the fibronectin gelatin-binding domain in a patient in need thereof.